Enzymes which act upon DNA or RNA, such as restriction endonucleases and exonucleases, ligases, polymerases, and others, have become widely available for use in research applications requiring modification, copying, and/or activation (e.g., transcription) of DNA. For example, restriction digests of DNA using specific endonucleases has aided in the recombinant expression of genes, and has been used for mapping and for cloning and sequencing DNA, as well as for the construction of specific probes for research and diagnostic applications.
Of interest to the present invention are numerous methods involving DNA or RNA modification which are improved upon by the invention. In the present application, methods for DNA or RNA modification include any method in which an enzyme, together with possible auxiliary proteins, acts upon DNA or RNA.
One of the most common methods involving DNA modification involves the digestion of DNA using restriction endonucleases. The most common restriction endonucleases cleave DNA at specific recognition sites within the DNA sequence. Sites at which a DNA sequence is interrupted (cleavage sites) are usually 4-8 base pairs long. Restriction digests are useful for the ultimate production of probes and for incorporation of fragments produced thereby into a vector for expression of a gene product or for cloning purposes. Especially where large amounts of DNA are to be digested, restriction digests may require large amounts of enzyme which are expensive and considerable time may be required for the reaction to run to completion.
Reactions involving DNA modification, such as those described above, are conventionally carried out in aqueous solution in the presence of organic and inorganic salts, buffers, and the required enzymes. The reaction mixtures must be incubated at optimized temperatures for set periods of time in order for the reaction to run to completion.
Also of interest to the present invention are lipids and their physical chemical properties. Lipids exhibit characteristic phase behavior which is a function of, inter alia, temperature and ionic concentrations. Lipid phase transitions are accompanied by dramatic alterations in physical organization. It is now established that several proteins bind to lipids in biological membranes and that such binding is a function of the phase state of the membrane lipids. By changing the physical state of the lipids in the bilayer, one may cooperatively regulate the binding, and therefore, the function of proteins which bind to the lipids. Specifically, several DNA-binding proteins have been shown to bind to lipids, with resulting changes in function depending upon the lipid phase state.
Phospholipids, for example, which are found in all cellular membranes, comprise two very different physical environments, a hydrophobic interior region and a more-complex hydrophilic exterior region. The hydrophobic tails of most phospholipids exist in two fundamental physical states. At higher temperatures the hydrophobic tails are in a fluid state and generally have rotational freedom; while at lower temperatures, phospholipids are more geometrically constrained. Overall, membrane lipids may exist in varying states of order, or phases, which normally depend upon both temperature and the lipid composition. There are four major forms of organization of lipids in a biological environment (i.e., in the presence of water). The lamellar liquid crystalline phase (L.sub..alpha.) is the fluid state normally depicted in representations of biological membranes. The lamellar gel phase (L.sub..beta.) is formed at low temperatures in lipids in which the lamellar structure is possible. The L.sub..beta. phase is characterized by tight packing and acyl chains which are more highly ordered as compared to the L.sub..alpha. phase. The L.sub..beta. phase is also characterized by a predominance of the all-trans acyl chain configuration, resulting in greater bilayer thickness than in the L.sub..alpha. phase. In the Hexagonal I phase (H.sub.I), lipids are organized in cylinder-like configurations, with the polar head groups facing outward. Finally, the Hexagonal II phase (H.sub.II) is characterized by a hexagonal array of cylinders, but with the polar head groups facing inward, surrounding a column of water (i.e., an inverted micelle). The phase adopted by a particular lipid is a function of, inter alia, temperature and the precise mix of lipids present. For example, some lipids, such as unsaturated phospatidylethanolamines, resist bilayer formation and tend toward the H.sub.II configuration. However, the overall configuration of a biological membrane is determined by the sum of phospholipids present, taking into account other factors, such as temperature, ionic strength, and hydration. The kinetic properties of membranes are due, in large part, to the properties of the phospholipids which comprise them. Membrane lipids create a fluid environment in which conformational changes in both membrane proteins and the lipids themselves allow a diversity of reactions to take place at the membrane which cause changes in cellular processes, including growth. The extent of fluidity in the membrane is a function of temperature, as indicated above, and also of the ratio of phospholipid to cholesterol and the extent of saturation of membrane lipids. One of the more important roles of membrane phospholipids occurs in the regulation of binding between membrane-bound receptors and their ligands.
The present invention provides materials and methods for enhancing the yield of restriction digestions, including enhancements in the amount of digestion production obtained per unit time, enhancements in the speed of digestion, enhancements in specificity, and reduced requirements for enzyme.